Pilot study of humanized glypican-3-targeted zirconium-89 immuno-positron emission tomography for hepatocellular carcinoma

This study


Background
Hepatocellular carcinoma (HCC) is increasing in incidence worldwide and has become the fastest growing cause of cancer death in the United States, with a median survival of less than 1 year [1][2][3].In order to improve survival with current treatments, HCC must be detected early when it is amenable to surgical resection or transplantation [3,4].However, multiphase, computed tomography (CT) or magnetic resonance imaging frequently misses lesions less than 1 cm, resulting in diagnostic uncertainty, delayed diagnosis, and early recurrence following resection [5][6][7].Innovative technology capable of detecting HCC with enhanced sensitivity and specificity is therefore imperative and pressing.
Our group previously demonstrated that immun-oPET using 89 Zr-labeled murine antibody targeting GPC3 ( 89 Zr-αGPC3 M ) reliably identified small HCCs in mice [6,10,11].Natarajan et al. described the use of 89 Zr-labeled humanized αGPC3 for HCC detection in a patient-derived xenograft model [16].We built on this important work by humanizing our radioimmunoconjugate (αGPC3 H ) and performing in vitro and novel in vivo comparisons to its murine predecessor.Here, we report that 89 Zr-αGPC3 H targets GPC3 comparably to 89 Zr-αGPC3 M , resulting in highly specific tumor uptake and successful HCC detection.

Methods
Creative Biolabs, Inc. (Shirley, NY) constructed αGPC3 H by engrafting of the parental murine antibody's complementarity-determining region (CDR).Flow cytometry was used to evaluate in vitro binding of αGPC3 M , a chimeric intermediary (αGPC3 C ), αGPC3 H , and αGPC3-deferoxamine (DFO) to HepG2 cells.Specifically, HepG2 cells were resuspended in cold FACS buffer and aliquoted at a concentration of 1 × 10 6 cells/100 μL.Primary and secondary antibodies were sequentially added to the cell suspensions and incubated at 4 °C protected from light for 30 min each.Cells were then washed, resuspended in cold buffer, and analyzed with the Symphony A3 (BD Biosciences, San Jose, CA) flow cytometer.A minimum of 10,000 cells were analyzed for each sample.Data analysis was performed using FlowJo software (TreeStar, Ashland, OR).
Orthotopic xenograft models of HCC were generated as previously described in athymic nude mice (Jackson Laboratories) [10][11][12]22].A final concentration of 1 × 10 6 HepG2 cells in 20 μL Geltrex (Gibco, Billings, MT) was injected into the left hepatic lobe.Two weeks after injection, bioluminescence imaging (BLI) was performed using the In Vivo Imaging System Spectrum (Perki-nElmer) to verify tumor establishment. Tmor size was calculated in a semi-quantitative manner based on mean photon emission (photons/sec) in a 2D region of interest (ROI) containing the tumor.
Humanized and murine αGPC3 were conjugated with eight equivalents of deferoxamine (DFO)-N-chlorosuccinimide and labeled with positron emitter 89 Zr as previously described [10].Isoelectric focusing was consistent with an average of less than one DFO moiety per antibody in both groups.The radiochemical yield of 89 Zr-DFO-αGPC3 H and 89 Zr-DFO-αGPC3 M was 98% and 89%, respectively.Radiochemical purity was determined by instant thin layer chromatography (iTLC) and confirmed by radioactive HPLC.Specific activity was calculated by dividing total radioactivity (GBq) of 89 Zr by total mg for each antibody.For simplicity, 89 Zr-DFO-αGPC3 is written as 89 Zr-αGPC3.
Mice were injected retro-orbitally with 8.1 to 10 megabecquerels (MBq) of 89 Zr-αGPC3 H (n = 11) or 89 Zr-αGPC3 M (n = 11).Five days after injection, mice with tumors predicted using BLI (n = 6 per group) underwent imaging using the Inveon small-animal PET/CT scanner (Siemens Medical Solutions USA, Inc.Molecular Imaging, Knoxville, TN).Horos software (Nimble Co., Annapolis, MD) was used for image analysis.Maximum activity concentration (MBq/mL) was measured in a 2D region of interest (ROI) to calculate tumor radioisotope uptake (percent injected dose per milliliter, %ID/mL), tumor-to-liver ratio, and tumor maximum standardized uptake value (SUV max ).
Biodistribution studies were performed separately in non-tumor-bearing, non-imaged mice 2 days after injection and in PET-imaged mice after imaging completion.
Ionization events (counts per minute, CPM) were measured for each organ and tumor (when applicable) using a Cobra II gamma counter (Packard Bioscience, Meriden, CT) and tissue uptake (%ID/g) was calculated.Livers from PET-imaged mice were processed for histopathology.Details provided in Supplementary Methods.

Humanized αGPC3 and αGPC3-DFO maintains GPC3 binding in vitro
Binding to HepG2 cell surface GPC3 by unconjugated αGPC3 M , αGPC3 C , and αGPC3 H was confirmed by flow cytometry (Fig. 1a).Binding of DFO-conjugated and αGPC3 M to GPC3 was overall similar to the unconjugated antibody (Fig. 1b).Binding of αGPC3 H and αGPC3 C to GPC3 was greater than αGPC3 M .

αGPC3 H is amenable to 89 Zr radiolabeling
The radiochemical purity of both 89 Zr-αGPC3 antibodies was > 98% and the specific activity was 0.14 GBq/mg.Details provided in Supplementary Methods.

Discussion
Humanized αGPC3 specifically targeted GPC3 in vitro and in vivo, enabling HCC detection with immunoPET in an orthotopic xenograft mouse model.This proofof-concept study builds on our prior research validating a murine radioimmunoconjugate for a theranostic approach to HCC, with potential to improve diagnosis, treatment, and survival [6,[10][11][12].
Our results demonstrate that humanization of 89 Zr-αGPC3 did not alter the highly avid binding to GPC3 on HepG2 cells and liver tumor xenografts.First, flow cytometry established at least equivalent, if not greater, binding of αGPC3 H to GPC3 compared with αGPC3 M , with minimal change when conjugated with DFO.Next, quality assurance of 89 Zr labeling confirmed that 89 Zr-αGPC3 H maintained high purity and specific activity.The majority of our experiments focused on the novel in vivo comparison between 89 Zr-αGPC3 H and 89 Zr-αGPC3 M .Five of six tumors in each group were detected by immunoPET, with no difference between groups in mean bioluminescence.Table 1 ImmunoPET and biodistribution data by mouse a Not measured due to minimal apparent signal b H6, M12: no tumor identified on PET/CT or histopathology (blank = not applicable) c PET and histologic findings suggest a tumor was present in H1, and the negative %ID/g was due to standard experimental error and a calculated baseline liver uptake that was higher than the measured tumor-containing left hepatic lobe uptake PET/CT data revealed no significant difference in mean tumor uptake and tumor-to-liver ratios (%ID/mL).Similarly, biodistribution analysis showed no difference in mean organ uptake, tumor uptake, and tumor-to-liver ratios (%ID/g).While finding comparability between 89 Zr-αGPC3 H and 89 Zr-αGPC3 M achieved the study's primary goal, additional details are worth noting.First, tumor uptake varied based on tumor size, with higher uptake in larger tumors as previously demonstrated [6].%ID/g (gamma counter) results were greater than %ID/mL (PET) due to limited PET/CT spatial resolution causing partial volume effect; hence, there could be a larger discrepancy between %ID/g and %ID/mL values in mice with smaller tumors (e.g., H3, M9) (Fig. 3, Table 1).While further consideration of the clinical impact of partial volume effect is warranted, this finding does highlight the successful detection of a range of tumor sizes with 89 Zr-αGPC3 immunoPET.Second, background liver uptake was greater in the 89 Zr-αGPC3 H group, which could imply Fc-mediated liver uptake of 89 Zr-αGPC3 H .However, our prior studies of mice injected with 89 Zr-αGPC3 M compared with non-GPC3targeting and GPC3-blocked controls demonstrated similar background liver uptake [6,11,16].Furthermore, the tumor-to-liver ratio by nature adjusts for such variables, with no difference between groups suggesting that tumor uptake was also proportionally higher in the humanized antibody group.In fact, tumor-to-liver ratios of 12 or greater indicate 89 Zr-αGPC3 H is highly specific for GPC3-expressing tumors [16].Finally, a limitation of our study is that we did not measure blood pool activity in biodistribution analyses and thus could not calculate tumor-to-blood ratios.However, we did include other highly vascular organs, and have shown in prior studies that 89 Zr-αGPC3 uptake in tumor and organs such as the liver, kidneys, and spleen is greater than blood pool activity beyond 24 h after injection [6,11].
Tumor presence was histopathologically confirmed in mice with PET-identified tumors, while no tumors were found on histologic analysis of livers without PETidentified tumors.A second limitation of our study is that, while meticulous gross examination of the liver and histopathologic analysis of suspected tumors was performed, serial sectioning of the entire left hepatic lobe was not undertaken due to funding constraints.Therefore, the discordance between BLI and PET for H6 and M12 is unresolved.Of note, the 3-week interval between imaging modalities was longer than in previous studies and thus tumor involution may have occurred.
Our study is similar to those from other groups in that it underscores the potential of human αGPC3 to detect HCC with immunoPET, however, there are key differences.Tumor-to-liver ratios by PET/CT and biodistribution analyses were notably higher than those reported by Natarajan et al. using a similar 89 Zr-labeled human αGPC3 IgG antibody and Fayn et al. using 89 Zr-labeled GPC3-targeting HN3 single-domain antibodies.In addition, there was a greater relative difference between tumor uptake and uptake in organs such as the heart, lungs, gastrointestinal tract, and kidneys on biodistribution analysis [16,17].While different methods for model development and radioimmunoconjugate injection used may affect the results such that they are not directly comparable [16,17], it is possible that our humanized antibody has a higher specificity for GPC3-expressing tumors.Furthermore, it should be noted that tumor-toliver ratios were measured 5 days after injection in this study compared with 1-7 days after injection in the aforementioned studies, however our prior experiments with 89 Zr-αGPC3 M demonstrated high tumor-to-liver ratios calculated from 4 h up to 7 days after injection [6,11].Finally, Carrasquillo et al. conducted a phase I clinical study of PET/CT in HCC patients using αGPC3 codrituzumab labeled with iodine-124 ( 124 I).While this valuable work underscores the clinical translatability of radiolabeled antibodies against GPC3, there was no tumor uptake in one patient and low tumor-to-liver ratios in several others [18].The authors stated that 89 Zr could have been a reasonable alternative to 124 I, and our findings support further investigation of 89 Zr-αGPC3 immunoPET to overcome challenges encountered with other radioimmunoconjugates.We appreciate the rigorous and ongoing work by our colleagues in the field and believe that parallel approaches to developing GPC3targeted radiolabeled imaging agents will be beneficial [14-20, 23, 24].

Conclusions
Humanized αGPC3 successfully targeted GPC3 in vitro and in vivo.Compared with our previously validated murine antibody, 89 Zr-αGPC3 H immunoPET demonstrated comparable HCC detection with highly specific tumor uptake in an orthotopic xenograft mouse model, affirming the efficacy and clinical translatability of 89 Zr-αGPC3 H immunoPET for HCC detection [16].Given our GPC3-targeted murine radioimmunoconjugates were previously validated for both immunoPET and cytotoxic RIT, immediate next steps include assessing treatment response using αGPC3 H -based RIT.This developing theranostic joins a growing field of other solid tumors, including colorectal, breast, prostate, renal cell cancers, non-Hodgkin's lymphoma, and neuroendocrine tumors, and has the potential to transform HCC management [10,25,26].

ROI Region of interest %ID/mL
Percent injected dose per milliliter SUV max Maximum standardized uptake value 124 I Iodine-124

Fig. 3 Fig. 3 (
Fig. 3 Humanized 89 Zr-αGPC3 immunoPET reliably identifies tumors.a Axial (top) and sagittal (bottom) PET/CT images of 89 Zr-αGPC3 H -and 89 Zr-αGPC3 M -injected mice.b Select H&E-stained liver sections.Blue arrowheads denote tumors.Scale bars 100 μm.c Tumor radioisotope uptake (%ID/mL).d Liver and tumor %ID/mL by mouse.e Tumor-to-liver ratio of %ID/mL; circle denotes largest tumors.f Tumor maximum standardized uptake (SUV max ).Each point or thin bar (d) denotes the value for each tumor-bearing mouse (n = 5/group); large bars denote the mean with SEM (See figure on next page.)